Mitochondrial DNA Restriction Fragment Length Research Articles

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.

Acanthamoeba: Keratopathogenicity of isolates from domestic tap water in Korea

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25 μg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69 μg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.

Genetic Relationships among Populations of the Senegalese Sole Solea senegalensis in the Southwestern Iberian Peninsula Detected by Mitochondrial DNA–Restriction Fragment Length Polymorphisms

AbstractWe assessed the population structure of the Senegalese sole Solea senegalensis in the southern Atlantic off the coast of the Iberian peninsula using restriction fragment length polymorphism analysis of three mitochondrial gene regions: 16S rRNA (620 base pairs [bp]), 12S rRNA (440 bp), and cytochrome b (380 bp). Fourteen composite haplotypes were detected among 109 adult fish from five natural populations. Haplotypic diversity values ranged between 0% and 18%, with the highest values in the Lisbon and Valdelagrana populations. A single composite haplotype predominated in most collections; two other haplotypes also occurred at low frequencies in three of the analyzed populations. The remaining 11 haplotyes were found only in single populations. A genetic differentiation index revealed significant differentiation between two groups (Lisbon and the other populations). However, from a conservative management perspective and based on the topology of the neighbor‐joining consensus tree and bootstrap values (54–88), the possibility of three groups (Lisbon, Portimao, and the other populations) should be considered.

Population Structure of Wild Musk Shrews (Suncus murinus) in Asia Based on Mitochondrial DNA Variation, with Research in Cambodia and Bhutan

The musk shrew (Suncus murinus) is a small mammalian species belonging to Insectivora. It is widely distributed in Asia. To identify the genetic relationship among wild musk shrew populations and examine its migration route, we investigated the populations of Cambodia and Bhutan by using mitochondrial DNA restriction fragment length polymorphism analysis and compared them with other Asian populations previously described. Four haplotypes were detected in Cambodia and eight in Bhutan. A total of 53 haplotypes were detected in Asia and were classified largely into two groups, the Continental and Island types, based on a minimum spanning network. From the distribution of mtDNA types in wild musk shrews, three major population groups are identified in Asia: South Asia, Southeast Asia, and Malay. It is suggested that the Malay population group was a mix of South and Southeast Asian population groups and that this was a contact area of the two groups. In addition, other contact areas between the South and Southeast Asian groups exist in Myanmar, but unlike the Malay, the Myanmar area was the border of these groups.

Clarification of the Etiology of Glomerella Leaf Spot and Bitter Rot of Apple Caused by Colletotrichum spp. Based on Morphology and Genetic, Molecular, and Pathogenicity Tests

ABSTRACT Morphological characteristics and vegetative compatibility groups (VCGs) of 486 isolates of Glomerella cingulata, Colletotrichum gloeosporioides, and C. acutatum collected from apple leaves with Glomerella leaf spot (GLS) symptoms and fruit with bitter rot symptoms in the United States and Brazil were studied. From this collection, 155 isolates of G. cingulata (93 from fruit, 61 from leaves, and 1 from buds), 42 isolates of C. gloeosporioides from fruit, and 14 isolates of C. acutatum (10 from fruit and 4 from leaves) were studied using mitochondrial (mt)DNA restriction fragment length polymorphism (RFLP) haplotypes. A subset of 24 isolates was studied by examining the sequence of a 200-bp intron of the glyceraldehyde 3-phosphate dehydrogenase (GDPH) nuclear gene. In addition, 98 isolates were tested for pathogenicity on leaves of cvs. Gala and Golden Delicious in the greenhouse, and 24 isolates were tested for pathogenicity on fruit of cv. Gala in growth chambers. In total, 238 and 225 isolates of G. cingulata were separated into four distinct morphological types and six VCGs, respectively. Five morphological types and six VCGs were identified among 74 and 36 isolates of C. gloeosporioides, respectively. Three morphological types and four VCGs were identified among 74 and 23 isolates of C. acutatum, respectively. Seven different mtDNA RFLP haplotypes were observed within isolates of G. cingulata, two within isolates of C. gloeosporioides, and two within isolates of C. acutatum. Phylogenetic trees, inferred based on maximum likelihood and maximum parsimony methods using the intron sequence, produced similar topologies. Each species was separated into distinct groups. All isolates tested were pathogenic on fruit, though only isolates with specific VCGs and haplotypes were pathogenic to leaves. Vegetative compatibility was a better tool than molecular characters for distinguishing isolates of G. cingulata pathogenic on both leaves and fruit from the ones pathogenic only on fruit. Isolates of G. cingulata capable of causing both GLS and bitter rot were included in haplotypes and groups based on the sequence analysis of the 200-bp intron that also included isolates capable of causing bitter rot only. Additionally, isolates of G. cingulata from the United States and Brazil which cause GLS were included in different haplotypes and sequence analysis groups. Therefore, one hypothesis is that isolates of G. cingulata from the United States capable of causing both GLS on foliage and bitter rot on fruit may have arisen independently of Brazilian isolates of G. cingulata capable of causing both GLS and bitter rot, and the two groups of isolates may represent distinct populations.

Sources of the adventitious microflora of a smear-ripened cheese

To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.

Survival of surface ripening cultures during storage and monitoring their development on cheese

To study the survival of bacteria isolated from the surface of smear cheese and monitor their development during cheese ripening. The storage of five potential bacterial surface-ripening cheese cultures, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variable, Microbacterium gubbeenense and Staphylococcus saprophyticus, in maximum recovery diluent (MRD), containing 0.85% w/v or 5% w/v NaCl, at 21 or 4 degrees C for 40 days, was investigated. All five strains studied survived well with a maximum decrease of c. 2.5 log(10) CFU ml(-1) after storage for 40 days at 4 degrees C in 0.85% or 5% w/v NaCl. Survival, especially of C. variable, was less at 21 degrees C. The development of defined ripening cultures containing C. casei and Debaryomyces hansenii on two farmhouse cheeses was also evaluated. Using pulsed-field gel electrophoresis (PFGE) for the bacteria and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) for the yeast, it was shown that the ripening cultures could be re-isolated in high numbers, 10(8) CFU cm(-2) for C. casei and 10(6) CFU cm(-2) for D. hansenii, from the cheese surface after 2.5 weeks of ripening. Ripening strains of surface ripening cultures can be stored in MRD containing 5% w/v salt at 4 degrees C for at least 40 days. Such cultures are recovered in high numbers from the cheese during ripening. This study has provided a low-cost and efficient way to store bacteria that could be used as ripening cultures for smear cheese. Such cultures can be recovered in high numbers from the cheese surface during ripening.

Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy ( Engraulis encrasicolus)

This study extends the geographic coverage of a previous study of mitochondrial DNA restriction fragment length polymorphism in European anchovy. Both studies together include 24 samples representing 17 localities extending from the Black Sea, through the Mediterranean Sea to the eastern Atlantic as far south as Dakar, Senegal. Eighty-eight haplotypes define two clades (A and B) separated by 3.2% sequence divergence. Clade A has a star-like genealogy indicative of a recent population expansion. Clade B has a more complex genealogy, consisting of several haplotypes at intermediate frequencies. The distributions of these clades consist of a mosaic with abrupt changes between some areas and gradients between other areas. Clade A predominates the Black and Aegean seas, but is present throughout the Mediterranean. Unexpectedly, new data show that clade A is also at a high frequency in the Atlantic, from Portugal to at least Senegal. Overall, the level of genetic differentiation among populations is high ( F ST = 0.148, p < 0.0001), with the greatest differences between basins. AMOVA reveals four main geographical groups: Atlantic, central Mediterranean, Aegean Sea, and Black Sea. Mismatch distribution clearly indicates historical bottleneck and population expansion for clade A, while for clade B such evidence is equivocal. This difference may reflect a range expansion for both clades, but with higher gene flow ( Nm values) between demes for clade A. Both contemporary and historical processes are important in shaping the complex genetic population structure of European anchovy.

Molecular characterization of Acanthamoeba isolated from amebic keratitis related to orthokeratology lens overnight wear

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patientos ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.

Surface Microflora of Four Smear-Ripened Cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.

Polymorphisms Among Chloroplast and Mitochondrial Genomes of Citrullus Species and Subspecies

Twelve and six DNA clones representing various parts of chloroplast and mitochondrial genomes, respectively, were used to detect polymorphism among five watermelon cultivars and 21 U.S. Plant Introductions (PIs) collected from diverse geographical locations and representing major groups of Citrullus species. Cluster analysis based on 20 chloroplast DNA (cpDNA) and 10 mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) markers differentiated the accessions into three major phenetic groups: PIs and watermelon cultivars of Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa (also designated as C. lanatus var. lanatus) (group I), PIs of C. lanatus var. citroides (of C. lanatus subsp. lanatus Schrad. ex Eckl. et Zeyh.)(group II), and C. colocynthis (L.) Schrad. PIs (group III). The chloroplast and mitochondrial genomes of watermelon cultivars are distinct, but closely related to those of the C. lanatus var. lanatus PIs. On the other hand, the chloroplast and mitochondrial genomes of the wild species C. colocynthis are more similar to those of C. lanatus var. citroides. Polymorphic cpDNA and mtDNA markers identified in this study can complement isozyme and nuclear DNA data used in earlier phylogenetic and phenetic classifications of Citrullus PIs. These cpDNA and mtDNA markers are being used in experiments designed to enhance watermelon cultivars by replacing the chloroplast and mitochondrial genome of cultivated watermelon with those of the wild species C. colocynthis.

Mitochondrial DNA diversity in 17th-18th century remains from Tenerife (Canary Islands)

Mitochondrial DNA sequences and restriction fragment length polymorphisms were retrieved (with >80% efficiency) from a 17th-18th century sample of 213 teeth from Tenerife. The genetic composition of this population reveals an important ethnic heterogeneity. Although the majority of detected haplotypes are of European origin, the high frequency of sub-Saharan African haplotypes (15.63%), compared to that of the present-day population (6.6%), confirms the importance of the Canary Islands in the black slave trade of that epoch. The aboriginal substrate, inferred from the U6b1 haplotypes (8.59%), has also decreased due to European input. Finally, the presence of Amerindian lineages (1.5%) reveals that the Canary Islands have also received genetic flow from America.

Comparison of AFLPs with other markers for phylogenetic inference in wild tomatoes [Solanum L. section Lycopersicon (Mill.) Wettst.

AbstractWild tomatoes (Solanum section Lycopersicon) are native to western South America. The delimitation and relationships of tomato species have differed widely depending upon whether morphological or biological species concepts are considered more important. Molecular data from mitochondrial, nuclear, and chloroplast DNA restriction fragment length polymorphisms (RFLPs), nuclear microsatellites, isozymes, and gene sequences of internal transcribed spacers of nuclear ribosomal DNA (ITS; multiple­copy), the single­copy nuclear encoded Granule­bound Starch Synthase gene (GBSSI or waxy), and morphology, have been used to examine hypotheses of species relationships. This study is a companion to the previous GBSSI gene sequence study and to the morphological study of relationships of all ten wild tomato species (including the recently described S. galapagense), with a concentration on the most widespread and variable species S. peruvianum s. l. These new AFLP data are largely concordant with the GBSSI and morphological data and in general support the species outlined in the latest treatment by C. M. Rick, but demonstrate the distinct nature of northern and southern Peruvian populations of S. peruvianum, and suggest that their taxonomy needs revision. Solanum ochranthum is supported as sister to wild tomatoes, and S. habrochaites and S. pennellii reside in a basal polytomy in the tomato clade.

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001–2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.

Genetic structure of korean wild populations of the Medaka Oryzias latipes inferred from allozymic variation.

Previous allozymic studies have revealed that Korean wild populations of Oryzias latipes have differentiated regionally, and are composed of two distinct groups, the East Korean Population and the China-West Korean Population. Recently, mitochondrial DNA (mtDNA) sequencing and restriction fragment length polymorphism (RFLP) analyses have confirmed these two groups, and shown that the distribution ranges of the two groups overlap in western Korea. In order to describe the detailed distributions of the two groups and the gene flow between them, genotypes of 13 allozymic loci were determined in 444 specimens from 96 localities in Korea. The two major groups were supported by remarkable allele frequency differences at six diagnostic loci: ACP*, AMY*, CK-A*, LDH-A*, PGM* and TF*. Individuals with the typical "eastern" genotype were mainly distributed in eastern and southern areas. In contrast, fish with the "western" genotype were predominant in the western area, and were further divided into two subgroups (the Han River and Geum River Subpopulations) by unique alleles at the ADH* locus. In the western coast, two distinct (eastern and western) genotypes were distributed in a mosaic fashion. This distribution pattern was identical to those from mtDNA analyses. Although the distribution patterns of the alleles at three loci (GPI-A*, LDH-C* and SOD*) showed introgressive conditions between the two groups, each population was nearly fixed as either the eastern or western genotype at all six diagnostic loci despite the proximity among samples. Therefore, it is suggested that some reproductive isolation mechanisms exist between the two groups in natural habitats.

Genetic diversity in farmed Asian Nile and red hybrid tilapia stocks evaluated from microsatellite and mitochondrial DNA analysis

We analyzed microsatellite and mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) in two domesticated (NIFI and Israel) and four genetically improved (GIFT, GMT, FAC-selected and SEAFDEC-selected) Nile tilapia ( Oreochromis niloticus) as well as five red hybrid tilapia ( Oreochromis mossambicus× O. niloticus) stocks (BFS, FACred, NIFIred, HL, and PF) farmed in Asia. Microsatellite variation at five loci ( UNH216, UNH172, UNH123, UNH147, UNH222) was more informative in characterizing stock differences than the mtDNA-RFLP markers that were based only on 14 restriction morphs. Contemporary microsatellite data showed that GIFT Nile tilapia had the highest mean expected heterozygosity ( H e=0.813), while GMT had the lowest ( H e=0.666). The unselected NIFI stock and SEAFDEC-selected were genetically similar, while GMT differed significantly from the other Nile tilapia stocks. Among the red tilapias, NIFIred had the highest H e (0.715), while BFS had the lowest variability ( H e=0.567). The Taiwanese red tilapia HL and Thai NIFIred were genetically similar. Except for NIFI, most of the Nile and red tilapia stocks exhibited remarkably significant homozygote excess relative to Hardy–Weinberg Equilibrium (HWE), suggesting some degree of inbreeding. Asian Nile tilapias were more genetically diverse (pooled H e=0.791; mtDNA nucleotide divergence value d A=0.009) than the red tilapias (pooled H e=0.697; mean d A=0.004). This slight divergence between the Nile and red tilapias was also seen in the analysis of molecular variance (AMOVA; F CT=0.0018) and in genetic distance and nucleotide divergence dendrograms. However, the AMOVA revealed that the greater percentage of variation (99.33%) in the total genetic diversity of the surveyed stocks is principally due to differences at the individual level and not between nor within groups. The significance of these results is that they reflect and lead to new inferences regarding the selective breeding and culture methods used in managing these farmed stocks.

Variation in the Population Structure of Yukon River Chum and Coho Salmon: Evaluating the Potential Impact of Localized Habitat Degradation

We used microsatellite and mitochondri-al DNA–restriction fragment length polymorphism (mtDNA–RFLP) analyses to test the hypothesis that chum salmon Oncorhynchus keta and coho salmon O. kisutch in the Yukon River, Alaska, exhibit population structure at differing spatial scales. If the hypothesis is true, then the risk of losing genetic diversity because of habitat degradation from a gold mine near a Yukon River tributary could differ between the two species. For each species, collections were made from two tributaries in both the Innoko and Tanana rivers, which are tributaries to the lower and middle Yukon River. The results revealed a large difference in the degree and spatial distribution of population structure between the two species. For chum salmon, the microsatellite loci (F-statistic (FST) = 0.021) and mtDNA (FST = −0.008) revealed a low degree of interpopulation genetic diversity on a relatively large geographic scale. This large-scale population structure should minimize, although not eliminate, the risk of genetic diversity loss due to localized habitat degradation. For coho salmon, the microsatellites (FST = 0.091) and mtDNA (FST = 0.586) revealed a high degree of interpopulation genetic diversity on a relatively small geographic scale. This small-scale population structure suggests that coho salmon are at a relatively high risk of losing genetic diversity due to localized habitat degradation. Our study underscores the importance of a multispecies approach for evaluating the potential impact of land-use activities on the genetic diversity of Pacific salmon.

Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom.

The incidence of acanthamoeba keratitis (AK) in the UK is some 15 times that in the United States and seven times that in Holland. To investigate reasons for this higher frequency, a study of the role of domestic tap water as a potential source of AK was undertaken. Tap outlets from the homes of 27 patients with culture-proven AK were sampled and cultured for free-living amoebae (FLA). For all Acanthamoeba isolates, mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) and cytochrome oxidase (cox 1/2) sequence typing was performed to determine the similarity between corneal and tap water isolates. FLA, including Acanthamoeba, were isolated from 24 (89%) of 27 homes, and the presence within the homes varied significantly with tap water temperature and location: 19 (76%) of 25 bathroom sink cold taps sampled compared with 6 (24%) of 25 hot and 9 (47%) of 19 kitchen cold taps compared with 3 (16%) of 19 of hot kitchen taps. Acanthamoeba were isolated from 8 (30%) of 27 homes (five bathroom sink cold taps, one cloakroom cold tap, one bath, and one bedroom sink mixer [hot/cold] taps). In six cases, identical Acanthamoeba mtDNA profiles were found for the clinical and home tap water isolates. In keeping with UK plumbing practice, 24 of 27 homes had internal roof water storage tanks to supply domestic taps, but the mains fed the kitchen cold tap. Water storage tanks promote colonization of domestic water with FLA, including Acanthamoeba, and hence increase the risk of AK. This accounts for the significantly greater incidence of AK in the UK and supports advice to avoid using tap water in contact lens care routines.

Isolates of Verticillium dahliae Pathogenic to Crucifers Are of at Least Three Distinct Molecular Types

ABSTRACT Diverse isolates of the soilborne wilt fungi Verticillium dahliae and V. albo-atrum were studied to understand the nature and origins of those infecting cruciferous hosts. All isolates from cruciferous crops produced microsclerotia, and the majority produced long conidia with a high nuclear DNA content; these isolates were divided into two groups by amplified fragment length polymorphism (AFLP) analysis. One group could be subdivided by other criteria such as rRNA sequences and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis. Two crucifer isolates were short spored and had a low nuclear DNA content. The results are consistent with the crucifer isolates being interspecific hybrids. The long-spored isolates are best regarded as amphihaploids (or allodiploids) with the AFLP groups probably each representing separate interspecific hybridization events. The short-spored crucifer isolates appear to be derived from interspecific hybrids and are here called 'secondary haploids'. Molecular evidence suggests that one parent in the crosses was similar to V. dahliae. The other parent of the amphihaploids seems to have been more similar to V. albo-atrum than to V. dahliae, but was distinct from all isolates of either species so far studied. The implications for the taxonomy of crucifer isolates are discussed and the use of the name V. longisporum, proposed elsewhere for just some of these isolates, is discouraged.

Analysis of mitochondrial DNA restriction fragment length polymorphisms for examining genetic variability among isolates of Phellinus linteus

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAs (mtDNAs) from nine Japanese wild isolates of Phellinus linteus was carried out to examine their genetic variability. BamHI and EcoRI digests of mtDNAs from these isolates produced four and five distinct RFLP patterns, respectively. By combining the RFLP patterns obtained with the two endonucleases, mtDNAs from the nine isolates could be assigned to five different genotypes, but no mtDNA variation was detected among the isolates collected from a small area. Distance values calculated among all pairs of mtDNA genotypes, based on the presence or absence of comigrating restriction fragments, were clearly smaller than those among the mtDNA genotypes of Lentinula edodes and Pleurotus ostreatus samples collected worldwide, suggesting the necessity of collecting P. linteus wild isolates for genetic resources from geographically wider areas.